Method of isolating and purifying keto steroids and new keto steroid compounds



METHOD OF ISOLATING AND PURIFYING KETO STOIiZJIgHIgS AND NEW KETOSTEROID COM- P 1) Leon Velluz, Paris, France, assignor to LesLaboratoires Francais de Chimiotherapie, Paris, France, a French bodycorporate No Drawing. Application April 20, 1956 Serial No. 579,457

Claims priority, application France December 11, 1952 8 Claims. (Cl.260--397.45)

The present invention relates to a process of purifying and isolatingketo steroid compounds and more particularly to an improved process ofselectively separating cortisone acetate from3,11,20-triketo-17a,21-dihydroxy pregnane.

The present invention is a continuation-in-part of my co-pendingapplication Serial No. 336,630, filed February 6, 1953, and entitledMethod of Isolating and Puritying Keto Steroids and New Keto SteroidCompounds.

It is known that a very important advance in the art was made bysuggesting to separate keto steroids from accompanying non-keto steroidsby reacting the keto compounds with ketone reagents. Two of the bestknown examples of such separation processes are the isolation of thesemicarbazone of trans-dehydroandrosterone, an important intermediateproduct in the synthesis of testosterone, from neutral cholesteroloxidation products, and the isolation of estrone from the urine ofpregnant mares by means of the reagents T and P of Girard andSandulesco. Reagents T and P permit the conversion of ketones into watersoluble compounds in the form of hydrazones with quaternary ammoniumresidues.

The semicarbazones, however, are practically almost insoluble and,therefore, do not permit any fractionation of the keto steroids. TheGirard and Sandulesco' reagents produce excellent results either onaccount of the different speed of reaction of various hormonestherewith, or on account of the difierences in hydrolysis of suchreaction products, or on account of the different pH- values requiredfor splitting up the hydrazones T or P into their corresponding ketocompounds on regeneration therefrom. But the working conditions withthese reagents are so delicate that their application is ratherrestricted and requires rather large volumes of liquids which arecumbersome to handle.

As stated above the semicarbazones are, in general, completelyinsoluble. This is also true with dinitro phenyl hydrazones which arepraised as being especially suitable for analytical purposes. Thesecompounds, however, can be hydrolized only with difficulty. The oximes,other classic keto derivatives, are usually too soluble.

The heretofore used ketone reagents have the further disadvantage thatthey do not possess sufficiently selective reactivity with various ketosteroids and that, therefore, they do not effect satisfactory separationof the most desired keto steroids.

It is one object of the present invention to provide a ketone reagentwhich allows selective precipitation of cortisone acetate from a mixtureof cortisone acetate and 3,11,20-triketo-l7a,2l-dihydroxy pregnane as itis obtained, for instance, when working up the mother liquors from themanufacture of cortisone.

Another object of the present invention is to provide a new ketonederivative of cortisone acetate which can readily be hydrolyzed tocortisone acetate.

Still another object of the present invention is to pro- 2,881,192Patented Apr. 7, 1959 vide a new ketone derivative of the acetate of3,11,20- triketo-17a,2l-dihydroxy pregnane.

Further objects of this invention will become apparent from thespecification and the examples given therein.

The present invention consists in principle in using as ketone reagentfor selectively precipitating reaction prodnets of said ketone reagentwith cortisone acetate from suitable starting materials and solutions,the hydrazide derived from diphenyl glycolic acid, also called benzilicacid. Said hydrazide will be designated herein and in the claims annexedhereto as reagent B. It corresponds to the following formula CoHtCortisone acetate reacts with said hydrazide according to the followingequation:

R /CaHa C=O+HzN-NHO CC R H 00H: keto steroid Benzillc acid hydrazidehormone Reagent B R\ Ca s C=NNH-O 0-0 1120 R H CaHu keto steroidhydrazone B Said hydrazone is only slightly soluble, and, therefore,

can readily be collected and separated from the reaction mixture, and iseasily split up, thereby regenerating the keto steroid componentthereof.

Benzilic acid hydrazide is a well known product and is producedaccording to the method of Curtius Journal der praktischen Chemie, 2ndseries, volume 95, page 196 (I917).

The hydrazone with cortisone acetate is produced, for instance, byreacting cortisone acetate or a mixture of cortisone acetate with otherketo steroids and more particularly a mixture of cortisone acetate withthe acetate of 3,11,20-triketo-l7u,2l-dihydroxy pregnane with saidbenzilic acid hydrazide in a suitable indifferent solvent and especiallya solvent of neutral reaction. It is advisable to add to the mixture asmall amount of an organic acid, such as acetic acid, which has acatalytic effect upon the reaction and accelerates the same. It is alsopossible to react the hydrazide and the keto steroid in pure aceticacid, i.e., without any indifferent solvent. Reaction takes place in thecold, after a more or less prolonged contact of the reaction components,or by heating the reaction mixture under reflux.

Reagent B reacts also with the acetate of3,11,21-triketo-17a,2l-dihydroxy pregnane and forms therewith ahydrazone of low solubility, for instance, in dichloro ethane but ofhigh solubility in methanol. After filtering off or centrifuging theprecipitated hydrazone B and subsequently recrystallizing saidhydrazone, it can be hydrolyzed to regenerate and recover the free ketosteroid compound. Such regeneration is usually effected by hydrolysiswith mineral acids or strong organic acids or by double decompositionand exchange with an aldehyde or a ketone which possesses a much greateraffinity to said reagent B than the keto steroid involved, whereby saidaldehyde or ketone is preferably used in excess of the required amountin order to cause a shift of equilibrium. Benzaldehyde or pyruvic acid,for instance, are

especially suitable for such exchange reaction, the latter" some acetateand that of the acetate of 3,11,20-triketo- 17a,2l-dihydroxy pregnane,the more so in view of the fact that the hydrazides of quite relatedacids, namely of mandelic acid and diphenyl acetic acid, do not showsuch selective solubility. The mandelic acid hydrazones are almost allsoluble under the usual reaction conditions when using methanol andethanol as solvents. The diphenyl acetic acid hydrazones, on the otherhand, do not show any differences in solubility; almost all of itshydrazones are insoluble so that no selective precipitation is achieved.

The process of reacting a keto steroid with reagent B according to thepresent invention may be employed with great advantage in themanufacture of cortisone. One step of the synthesis of this compoundconsists in oxidizing 11,20-diketo-3a,17a-dihydroxy-2l-acetoxy pregnaneto 3,11,20-triketo-17a-hydroxy-21-acetoxy pregnane. It was found thatthe small amounts of unoxidized starting material considerably disturband impede the following reaction steps. It was found that purificationby recrystallization does not yield satisfactory results and does notremove said impurity. This problem, however, is readily solved byemploying the hydrazide of benzilic acid as re agent. With said reagentunoxidized 11,20-diketo-3a,l7adihydroxy-Zl-acetoxy pregnane forms ahydrazone B which is readily soluble, for instance, in methanol whilethe hydrazone B of its triketo oxidation product is remarkably insolubletherein.

Hydrazone B of cortisone acetate of the following formula is soluble indichloro ethane while the hydrazone B of the 4,5-saturated cortisoneacetate of the following formula HO O O-GHzOJS 0.011

CsHs

o-o -HN-N is not at all soluble therein.

One may proceed, without in anyway departing from the principles of thisinvention, in such a manner that first the mixture of keto steroids isreacted with reagent B in a solvent wherein all the hydrazones B aresoluble, whereafter one of the hydrazones B is precipitated by addinganother solvent to the solution wherein said particular hydrazone B tobe separated is insoluble. One may, of course, also evaporate to drynessthe solution of the hydrazones B in a solvent wherein all of them aresoluble, and may extract from the evaporation residue by means ofanother suitable solvent the one or the other of said hydrazones B. Incases in which the desired product contains only one impurity, one mayreact said impurity with reagent B to eliminate the same.

The following examples serve to illustrate this invention and some ofthe advantageous features thereof. will appear from the descriptionofpreferred modes of carrying out the invention. It is to be understood,of course, that the invention is not limited to the precise order ofstepsof thev processes, setforth, as the, invention can be a 4 embodiedin a plurality and a variety of forms and can be practiced in aplurality and a variety of ways.

Thus one may use other indifferent solvents than those mentioned in theexamples, such as isopropanol, dioxane, benzene, as well as otherorganic acids, alone or mixed, such as formic or tartaric acid.

The melting points given in the following examples were determined inthe Maquenne block and indicate the point of instantaneous melting.

EXAMPLE I Hydrazone B of cortisone acetate and regeneration of cortisoneacetate therefrom.

50 g. of cortisone acetate (3,11,20-n-iketo-l7a-hydroxy- 21-acetoxy-A-pregnene) and 33 g. of reagent B are boiled under reflux in 1 liter ofmethanol containing 10 cc. of acetic acid. After cooling the reactionmixture, the precipitated crystals are filtered off, washed, and dried.The yield is 96%. The benzilic acid hydrazone of cortisone acetatecrystallizes in platelets which are insoluble in ether and only slightlysoluble in chloroform and acetic acid. Melting point: 196 C.; specificrotatory power: [011 +235 to 240 (in 1% chloroform solution).

Said hydrazone B is hydrolyzed in the following manner:

50 g. of said hydrazone B are treated with a mixture of 200 cc. ofglacial acetic acid, 20 cc. of pyruvic acid, and 10 cc. of water. Thecrystals obtained thereby are filtered off and washed first with asolution of 5% sodium bicarbonate and then with water. The washedproduct is dried. 30 g. of cortisone acetate are obtained. Meltingpoint: 247 C.; specific rotatory power [a] ;+177 (in 1% acetonesolution). Yield:

EXAMPLE 2 Purification of cortisone acetate 40 g. of impure cortisoneacetate, melting at 230 C., are heated under reflux with 30 g. ofreagent B in 1,000 cc. of methanol containing 1% of acetic acid. Oncooling, hydrazone B of cortisone acetate melting at 196 C. and having aspecific rotatory power of [a] :-|-23524O (in 1% chloroform solution),is obtained.

Said hydrazone B is hydrolyzed in the same manner as described inExample 1, thereby yielding cortisone acetate melting at 247 C. andhaving a specific. rotatory power [a] :+177 (in 1% acetone solution).

EXAMPLE 3 Hydrazone B of 3,11,20-triketo-17u-hydr0xy- 21-acet0xypregnane g. of reagent B and 100 g. of3,11,20-triketo-l7ahydroxy-21-acetoxy pregnane are heated under refluxin 200 cc. ofdichloro ethane. The reaction mixture is cooled, theprecipitated crystals are filtered off, washed, and dried. g. of thebenzilic acid hydrazone of 3,1l,ZO-triketo-17a-hydroxy-21-acetoxypregnane are obtained corresponding to a yield of 95%. Said hydrazone Bforms needles which melt at 210 C. and have a specific rotatory power of[a] :+1O1 (in 1% pyridine solution). Yield: 95 The hydrazone B is onlyslightly soluble in the ordinary solvents but is soluble in pyridine.

Said hydrazone B is decomposed in the following manner: 50 g. thereofare treated with a mixture of 200 cc. of glacial acetic acid, 20 cc. ofpyruvic acid, and 10 cc. of water. The crystals formed thereby arewashed with a 5% solution of sodium bicarbonate, subsequently with Nhydrochloric acid, and finally with water. After drying, 30 g.,corresponding to 95%, of 3,11,20-triketol7a-hydroxy-2l-acetoxy pregnanehaving a melting point of 231 C. and a specific rotatory power of [a]:+82 (in 1% acetonesolution), are. obtained thereby.

EXAMPLE 4 Purification of 3,11,20-triket-1fizz-hydroxy- ZI-aeetoxypregnane 20 g. of 3,11,20-triketo-17a-hydroxy-21-acetoxy pregnanecontaining, as impurity, 20% of 11,20-dik6tO-3oz,17u-dihydroxy-21-acetoxy pregnane are boiled under reflux in 350 cc. ofmethanol containing 1% of acetic acid, with 19 g. of reagent B. Oncooling, 23 g. of crystals melting at 210 C. are obtained whichrepresent the hydrazone B of the triketo compound. Hence, the yield is95%.

Said hydrazone B is decomposed as described in Example 1 whereby pure3,11,20-triketo-l7a-hydroxy-21- acetoxy pregnane melting at 231 C. andhaving a specific rotatory power [m] :-|82 (in 1% acetone solution) isobtained.

EXAMPLE Separation of cortisone acetate from the 21-acetate of3,11,20-triketo-17a,21-dihydroxy pregnane 43 g. of a residue obtained onevaporation of the mother liquors from the known cortisone manufacturingprocesses, which residue contains 3,11,20-tn'keto-17a,21-dihydroxypregnane, are acylated by means of acetic acid anhydride and pyridine.The reaction product, which forms a yellowish gum, is dissolved, whileheating to boiling, in 300 cc. of methanol containing 1% of acetic acid.A boiling solution of 26 g. of reagent B (excess of in 200 cc. ofmethanol containing 1% of acetic acid is added thereto and the mixtureis heated under refiux on a water bath. The hydrazones B crystallizethereby. The mixture is allowed to stand overnight. The crystalline massis centrifuged, washed with methanol, thereafter with N hydrochloricacid to eliminate the excess of reagent B, and finally with water, andis dried at 120 C. 45.8 g., corresponding to 68.5%, of a mixture ofhydrazones B melting at about 200-210 C. are obtained.

Said 45.8 g. of hydrazones B are mixed with 200 cc. of dichloro ethane.The mixture is kept lukewarm on the water bath for several minutes andis then cooled to room temperature. The insoluble material is removed byfiltration and is again mixed with 200 cc. of dichloro ethane. Themixture is treated as described above and the whole procedure is againrepeated with 50 cc. of dichloro ethane. On drying, 26 g. of hydrazone Bof 3,1l,ZO-triketo-17u-hydroxy-21-acetoxy pregnane melting at 210 C.,are obtained, corresponding to about 56.5% of the mixture used asstarting material.

The dichloro ethane extracts are combined and distilled to dryness in avacuum. 18.5 g., corresponding to 40%, of hydrazone B of cortisoneacetate are obtained in crystalline form on addition of aqueous aceticacid.

On splitting up said two hydrazones B according to the methods describedin the preceding examples, on the one hand, cortisone acetate, and, onthe other hand, 3,11,20-triketo-17a-hydroxy-2l-acetoxy pregnane areregenerated.

In place of cortisone acetate, other esters with lower alkanoic acidsmay also be used as starting material.

Of course, many changes and variations in the reaction conditions, thesolvents used, the temperature and duration of the condensationreaction, the methods of isolating, purifying, and working up thehydrazones B, the manner in which said hydrazones B are decomposed andsplit up to the corresponding ketones and the like may be made by thoseskilled in the art in accordance with the principles set forth hereinand in the claims annexed hereto.

I claim:

1. The 3-mono-benzilic acid hydrazone of a keto steroid compoundselected from the group consisting of the ester of cortisone with alower alkanoic acid and the ester of 3,11,20-triketo-17a,21-dihydroxypregnane with a lower alkanoic acid.

2. The 3-mono-benzilic acid hydrazone of cortisone acetate.

3. The 3-mono-benzilic acid hydrazone of3,11,20-t1'iketo-l7a-hydroxy-2l-acetoxy pregnane.

4. In a process of purifying and isolating cortisone acetate from the21-acetate of 3,11,20-triketo-17a,21-dihydroxy pregnane, the stepscomprising dissolving said mixture in methanol containing acetic acid,adding thereto the hydrazide of benzilic acid, boiling the reactionmixture under reflux until condensation is completed, cooling thereaction mixture, separating the precipitated mixture of hydrazones B,mixing and moderately heating said mixture with dichloro ethane andrepeating said mixing and moderately heating of the undissolvedhydrazones B with dichloro ethane until no more hydrazone is dissolved,separating the undissolved hydrazone B of 3,11,20-triketo'17a-hydroxy-21-acetoxy pregnane from the dichloro ethane solutions, andremoving the solvent from the combined dichloro ethane extracts, therebyyielding the hydrazone B of cortisone acetate.

5. In a process of purifying and isolating cortisone esters with loweralkanoic acids from a mixture containing such cortisone ester and the21-ester of the correspond ing pregnane compound, the steps comprisingadding benzilic acid hydrazide to a solution of said esters in methanol,permitting said benzilic acid hydrazide to condense with said esters,extracting the hydrazone B of the cortisone ester from the resultingmixture of hydrazones B by means of dichloro ethane, isolating thehydrazone B of the cortisone ester from the dichloro ethane extract, andsplitting up the resulting benzilic acid hydrazone of the cortisoneester to the substantially pure cortisone ester.

6. The process of purifying and isolating cortisone esters according toclaim 5, wherein condensation of the mixture of cortisone esters and theesters of the corresponding pregnane compound is carried out whileheating.

7. The process of purifying and isolating cortisone esters according toclaim 5, wherein condensation is carried out in the presence of an acidagent.

8. The process of purifying and isolating cortisone esters according toclaim 7, wherein the acid agent is acetic acid.

References Cited in the file of this patent UNITED STATES PATENTS2,062,904 Hartmann Dec. 1, 1936 2,103,735 Schoeller Dec. 28, 19372,188,881 Butenandt Jan. 30, 1940 OTHER REFERENCES Fieser et al.:Natural Products Related to Phenanthrene, 3rd edition, pages 308-309,405-406 (1939).

1. THE 3-MONO-BENZILIC ACID HYDRAZONE OF A KETO STERIOD COMPOUNDSELECTED FROM THE GROUP CONSISTING OF THE ESTER OF CORTISONE WITH ALOWER ALKANOIC ACID AND THE ESTER OF 3,11,20-TRIKETO-17A,21-DIHYDROXYPREGANE WITH A LOWER ALKANOIC ACID.
 4. IN A PROCESS OF PURIFYING ANDISOLATING CORTISONE ACETATE FROM 21-ACETATE OF3,11,20-TRIKETO-17A,21-DIHYDROXY PREGANE, THE STEPS COMPRISINGDISSOLVING SAID MIXTURE IN METHANOL CONTAINING ACETIC ACID, ADDINGTHERETO THE HYDRAZIDE OF BENZILIC ACID, BOILING THE REACTION MIXTUREUNDER REFLUX UNTIL CONDENSATION IS COMPLETED, COOLING THE REACTIONMIXTURE, SEPARATING THE PRECIPITATED MIXTURE OF HYDRAZONES B, MIXING ANDMODERATELY HEATING SAID MIXTURE WITH DICHLORO ETHANE AND REPEATING SAIDMIXING AND MODERATELY HEATING OF THE UNDISSOLVED HYDRAZONES B WITHDICHLORO ETHANE UNTIL NO MORE HYDRAZONE IS DISSOLVED, SEPARATING THEUNDISSOLVED HYDRAZONE B OF 3,11,20-TRIKETO17A-HYDROXY-21-ACETOXY PREGANEFROM THE DICHLORO ETHANE SOLUTIONS, AND REMOVING THE SOLVENT FROM THECOMBINED DICHLORO ETHANE EXTRACTS, THEREBY YIELDING THE HYDRAZONE B OFCORTISONE ACETATE.